基于叶绿体DNA变异的山荆子种质遗传多样性和系统演化

doi:10.3864/j.issn.0578-1752.2020.03.012

摘要/Abstract

摘要：

【目的】山荆子是中国原产苹果属植物中分布最广泛的种,母系遗传的叶绿体基因组的非编码区适用于较低的分类阶元（如科、属）的系统研究。对野外考察新收集的山荆子种质的叶绿体DNA（cpDNA）非编码区进行测序,解析其序列遗传变异,从母系遗传基因的角度探究山荆子不同居群的遗传多样性和系统演化关系,为我国山荆子种质资源的起源演化以及收集和保护提供理论依据。【方法】利用4对叶绿体DNA引物扩增新收集的215份山荆子种质资源的4个非编码区trnH-psbA、trnS-trnG spacer+intron、trnT-5'trnL和5'trnL-trnF,对每个基因间区正反向测序获得的序列进行人工校对后,使用MEGA 7.0进行序列拼接和比对,并构建山荆子不同居群间基于遗传距离的Neighbour-Joining系统发育树;使用DnaSP ver5.10.01计算叶绿体DNA的遗传多样性参数,计算不同居群间的基因流和基因分化;利用Arlequin v3.5分析标准分子变异（AMOVA）;运用NetWork ver4.6.1.2构建种内居群间的叶绿体DNA单倍型邻接网络关联图。【结果】4个叶绿体DNA非编码区经测序、拼接、比对和合并之后的片段长度为3 777 bp,共有171个多态性变异位点,其中包含150个插入-缺失位点、20个简约信息位点和1个单一突变位点。在215份山荆子种质中,trnH-psbA、trnS-trnG spacer + intron、trnT-5'trnL和5'trnL-trnF区域的变异位点数量分别为26、32、103和10个,单倍型数量分别为8、8、6和4个,合并之后的叶绿体DNA片段的单倍型为24个。核苷酸多样性最高的区域为trnT-5'trnL（Pi=0.01174）,单倍型（基因）多样性最高的为trnS-trnG spacer+intron（Hd=0.599）,最低的为5'trnL-trnF（Hd=0.228）。215份山荆子种质叶绿体DNA多样性较高（Hd=0.727,Pi=0.00577）。Tajima’s D检验中,4个cpDNA区域在各检验水平上均不显著,检测的4个cpDNA区域在进化上遵循中性模型。AMOVA分析表明遗传变异主要存在于群体内部。【结论】供试4个叶绿体DNA非编码区适合苹果属山荆子种质遗传多样性和系统演化分析。在叶绿体DNA水平导致山荆子群体进化的原因不是自然选择,而是突变压力和遗传漂变。群体间遗传分化与其地理距离不完全相关。山荆子可能为多点起源,推测黑龙江和吉林,内蒙古,甘肃和山西为3个可能的起源地区。

关键词: 山荆子, 叶绿体DNA, 非编码区, 遗传多样性, 系统演化

Abstract:

【Objective】Malus baccata (L.) Borkh is the most widely distributed native species in China. The non-coding region of chloroplast genome by maternal inheritance is suitable for the systematic study of lower taxonomic levels (such as families and genera). The non-coding regions of cpDNA of 215 germplasms were sequenced, and their genetic variation was analyzed. In this study, the genetic diversity of Malus baccata and the phylogenetic relationship among different populations were explored from the perspective of maternal inheritance, which provided a theoretical basis for origin and genetic evolution, collection and protection of Malus baccata germplasm resources in China.【Method】Four non-coding regions (trnH-psbA, trnS-trnG spacer + intron, trnT-5'trnL and 5'trnL-trnF) of 215 germplasms were amplified by four primers. After manually proofreading sequences obtained through forward and backward sequencing, MEGA 7.0 was used for sequence splicing and alignment, and based on genetic distance, the Neighbour-Joining phylogenetic tree was constructed among different populations of Malus baccata. DnaSP ver5.10.01 was used to calculate the genetic diversity parameters of chloroplast DNA, gene flow and gene differentiation among different populations. Arlequin v3.5 was used to analyze standard molecular variation (AMOVA), and NetWork 4.6.1.2 was used to construct Median-Joining network for cpDNA haplotypes among intraspecific populations of Malus baccata.【Result】The length of four non-coding regions of chloroplast DNA was 3 777 bp after sequencing, splicing, alignment and merging, and 171 variable sites were detected, which included 1 singleton variable sites, 20 parsimony informative sites and 150 insertion-deletion gaps. Among 215 accessions of Malus baccata, the number of variable sites of region trnH-psbA, trnS-trnG spacer + intron, trnT-5'trnL and 5'trnL-trnF were 26, 32, 103 and 10, respectively. The number of haplotypes for four regions were 8, 8, 6 and 4, respectively, and after four regions merged, the haplotypes of chloroplast DNA fragments were 24. The region with highest nucleotide diversity was trnT-5'trnL (Pi=0.01174), and the region with highest haplotype diversity was trnS-trnG spacer + intron (Hd=0.599), and the haplotype diversity of 5'trnL-trnF was the lowest (Hd=0.228). The cpDNA diversity of Malus baccata was high (Hd=0.727, Pi=0.00577). Tajima’s test showed all Tajima’s D values were not statistical at different levels, which indicated that variation of those chloroplast regions followed natural theory of molecular evolution. AMOVA showed that genetic variation mainly existed within populations.【Conclusion】The four non-coding regions of chloroplast DNA were suitable for the analysis of genetic diversity and phylogenetics of Malus baccata. At the cpDNA level, it was not natural selection, but mutation pressure and genetic drift that led to population evolution of Malus baccata. The genetic differentiation among populations was not completely correlated with their geographical distance. Malus baccata might originate from several sites, and the three possible origins, including Heilongjiang and Jilin, Inner Mongolia, Gansu and Shanxi, were inferred.

Key words: Malus baccata, chloroplast DNA, non-coding region, genetic diversity, phylogenetics

高源,王大江,王昆,丛佩华,张彩霞,李连文,朴继成. 基于叶绿体DNA变异的山荆子种质遗传多样性和系统演化[J]. 中国农业科学, 2020, 53(3): 600-611.

GAO Yuan,WANG DaJiang,WANG Kun,CONG PeiHua,ZHANG CaiXia,LI LianWen,PIAO JiCheng. Genetic Diversity and Phylogenetics of Malus baccata (L.) Borkh Revealed by Chloroplast DNA Variation[J]. Scientia Agricultura Sinica, 2020, 53(3): 600-611.

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编号 Code	cpDNA间区 cpDNA intergenic regions	F序列（5′-3′） Forward sequence (5′-3′)	R序列（5′-3′） Reverse sequence (5′-3′)	扩增片段长度 Amplified fragment length
CP20	trnH-psbA	CGCGCATGGTGGATTCACAATCC	GTTATGCATGAACGTAATGCTC	~400
CP21	trnS-trnG spacer + intron	AGATAGGGATTCGAACCCTCGGT	GTAGCGGGAATCGAACCCGCATC	~1500
CP22	trnT-5' trnL	CATTACAAATGCGATGCTCT	TCTACCGATTTCGCCATATC	~1200
CP23	5' trnL-trnF	ATTTGAACTGGTGACACGAG	CGAAATCGGTAGACGCTACG	~1000

编号 Code	扩增区域 Region	PCR反应条件 PCR condition	退火温度 Annealing temperature
CP20	trnH-psbA	80℃ 5 min;94℃ 30 s,56℃ 30 s,72℃ 1 min,35个循环（35 cycles）;72℃ 10 min	56℃
CP21	trnS-trnG spacer + intron	80℃ 5 min;96℃ 10 s,50℃ 5 s,60℃ 4 min,30个循环（30 cycles）;60℃ 10 min	50℃
CP22	trnT-5' trnL	96℃ 5 min;96℃ 1 min,57℃ 2 min,72℃ 2.5 min,34个循环（34 cycles）;72℃ 10 min	57℃
CP23	5' trnL-trnF	80℃ 5 min;96℃ 10 s,50℃ 5 s,60℃ 4 min,30个循环（30 cycles）;60℃ 10 min	50℃

叶绿体DNA区域 cpDNA regions	片段长度 Length of fragment (bp)	变异位点 Variable sites (Vs)	单一突变位点 Singleton variable sites (Ss)	简约信息位点 Parsimony informative sites (Ps)	插入-缺失位点 Insertion-deletion gaps (Is)	核苷酸多样性 Nucleotide diversity (Pi)	平均核苷酸差异 Average number of nucleotide difference (K)
trnH-psbA	285	26	0	2	24	0.00913	3.13236
trnS-trnG spacer+intron	1383	32	1	5	26	0.00311	4.52649
trnT-5' trnL	1140	103	0	11	92	0.01174	15.68937
5' trnL-trnF	965	10	0	2	8	0.00045	0.44216
组合 Combined	3773	171	1	20	150	0.00577	23.79039

cpDNA区域 cpDNA Regions	单倍型数目 Number of haplotypes (h)	单倍型（基因）多样性 Haplotype (Gene) diversity (Hd)	单倍型多样性方差 Variance of haplotype Diversity (Vh)	单倍型多样性标准差 Standard deviation of haplotype diversity (Sh)
trnH-psbA	8	0.4762	0.00159	0.040
trnS-trnG spacer + intron	8	0.599	0.00111	0.033
trnT-5' trnL	6	0.372	0.00168	0.041
5' trnL-trnF	4	0.228	0.00133	0.037
组合 Combined	24	0.727	0.00084	0.029

cpDNA区域 cpDNA regions	Tajima's D值 Tajima's D	显著性 Significance
trnH-psbA	-0.78313	P>0.10
trnS-trnG spacer+ intron	-0.45018	P>0.10
trnT-5' trnL	-0.29083	P>0.10
5' trnL-trnF	-1.70516	0.05<P<0.10
组合 Combined	-0.54177	P>0.10